Acetylcholinesterase is the enzyme responsible for the hydrolysis of acetylcholine, while nicotinic acetylcholine receptors contain cation-selective channels that open in response to binding acetylcholine. These two proteins play central roles in cholinergic neurotransmission, and they are therefore essential for normal neuronal function. Drugs that affect their activity are widely used as insecticides, as adjuvants in surgical anesthesia, and as therapeutic agents for the treatment of glaucoma, paralytic ileus, atony of the urinary bladder, and myasthenia gravis. All of the clinically useful drugs that affect the activity of these proteins do so by interacting with their acetylcholine-recognition sites. Therefore, structural and pharmacological characterization of these sites is essential for understanding the function of these proteins and for developing new and more useful drugs. The research proposed herein is directed towards the structural and functional characterization of the acetylcholine-recognition sites of acetylcholinesterase and the nicotinic acetylcholine receptor. In the first stage of the project an emphasis will be placed on using small, covalent, active-site directed affinity reagents to directly identify amino acids within the acetylcholine-binding sites. Examples include synthetic active-site directed crosslinking reagents, and the naturally occurring toxins onchidal and lophotoxin. (Onchidal and lophotoxin are active-site directed covalent inhibitors of acetylcholinesterase and nicotinic acetylcholine receptors, respectively). In the second stage of the project the amino acids identified with the active-site directed affinity reagents will be altered by site-directed mutagenesis and their function will be assessed by heterologous expression. Integration of experimental pharmacology, medicinal chemistry, protein chemistry, and molecular biology will increase our understanding of the molecular mechanisms by which these important proteins recognize and bind acetylcholine. As a result, such studies will ultimately aid in the design of new and more useful drugs and insecticides.